Maternal gene expression profiling during pregnancy and preeclampsia in human peripheral blood mononuclear cells.

UNLABELLED: Preeclampsia is a major obstetrical complication affecting maternal and fetal health. While it is clear that there is a substantial placental contribution to preeclampsia pathogenesis, the maternal contribution is less well characterized. We therefore performed a genome-wide transcriptome analysis to explore disease-associated changes in maternal gene expression patterns in peripheral blood mononuclear cells (PBMCs). METHODS: Preeclampsia was defined as gestational hypertension, proteinuria and hyperurecimia. Total RNA was isolated from PBMCs obtained from women with uncomplicated pregnancies (n = 5) and women with preeclamptic pregnancies (n = 5). Gene expression analysis was carried out using Agilent oligonucleotide microarrays. Biological pathway analysis was undertaken using Ingenuity Pathway Analysis software. Quantitative real-time PCR (QRTPCR) was performed to validate the gene expression changes of selected genes in normotensive and preeclamptic patients (n = 12 each). RESULTS: We identified a total of 368 genes that were differentially expressed in women with preeclampsia compared to normal controls with false discovery rate (FDR) controlled at 10%. In follow up experiments we further analyzed the expression levels of a number of genes that were identified as altered by the microarray data including survivin (BIRC5), caveolin (CAV1), GATA binding protein-1 (GATA1), signal tranducer and activator of transcription 1 (STAT1), E2F transcription factor-1 (E2F1), fibronectin-1 (FN1), interleukin-4 (IL-4), matrix metalloprotease-9 (MMP-9) and WAP four disulfide domain protein (WFDC-1) by QRTPCR. Additionally we performed immuno blot analysis and zymography to verify some of these candidate genes at the protein level. Computational analysis of gene function identified an anti-proliferative and altered immune function cellular phenotype in severe preeclamptic samples. CONCLUSIONS: We have characterized the genome-wide mRNA expression changes associated with preeclampsia-specific genes in circulating maternal blood cells at the time of delivery. In addition to providing information relating to the biological basis of the preeclampsia phenotype, our data provide a number of potential biomarkers for use in the further characterization of this disease.

Comparative SAGE analysis of the response to hypoxia in human pulmonary and aortic endothelial cells.

We utilized serial analysis of gene expression (SAGE) to analyze the temporal response of human pulmonary artery endothelial cells (HPAECs) to short-term chronic hypoxia at the level of transcription. Primary cultures of HPAECs were exposed to 1% O2 hypoxia for 8 and 24 h and compared with identical same-passage cells cultured under standard (5% CO2-95% air) conditions. Hierarchical clustering of significant hypoxia-responsive genes identified temporal changes in the expressions of a number of well-described gene families including those encoding proteins involved in thrombosis, stress response, apoptosis, angiogenesis, and cell proliferation. These experiments build on previously published data describing the transcriptomic response of human aortic endothelial cells (HAECs) obtained from the same donor and cultured under identical conditions, and we have thus taken advantage of the immortality of SAGE data to make direct comparisons between these two data sets. This approach revealed comprehensive information relating to the similarities and differences at the level of mRNA expression between HAECs and HPAECs. For example, we found differences in the cell type-specific response to hypoxia among genes encoding cytoskeletal factors, including paxillin, and proteins involved in metabolic energy production, the response to oxidative stress, and vasoreactivity (e.g., endothelin-1). These efforts contribute to the expanding collection of publicly available SAGE data and provide a foundation on which to base further efforts to understand the characteristics of the vascular response to hypoxia in the pulmonary circulation relative to systemic vasculature.

Genome-wide analysis of the endothelial transcriptome under short-term chronic hypoxia.

We have utilized serial analysis of gene expression (SAGE) to analyze the temporal response of human aortic endothelial cells (HAECs) to short-term chronic hypoxia at the level of transcription. Primary cultures of HAECs were exposed to 1% O2 hypoxia for 8 and 24 h and compared with identical same passage cells cultured under standard (5% CO2-95% air) conditions. A total of 121,446 tags representing 37,096 unique tags were sequenced and genes whose expression levels were modulated by hypoxia identified by novel statistical analyses. Hierarchical clustering of genes displaying statistically significant hypoxia-responsive alterations in expression revealed temporal modulation of a number of major functional gene families including those encoding heat shock factors, glycolytic enzymes, extracellular matrix factors, cytoskeletal factors, apoptotic factors, cell cycle regulators and angiogenic factors. Within these families we documented the coordinated modulation of both previously known hypoxia-responsive genes, numerous genes whose expressions have not been previously shown to be altered by hypoxia, tags matching uncharacterized UniGene entries and entirely novel tags with no UniGene match. These preliminary data, which indicate a reduction in cell cycle progression, elevated metabolic stress and increased cytoskeletal remodeling under acute hypoxic stress, provide a foundation for further analyses of the molecular mechanisms underlying the endothelial response to short-term chronic hypoxia.

Genomic analysis of immediate/early response to shear stress in human coronary artery endothelial cells.

The involvement of shear stress in the pathogenesis of vascular disease has motivated efforts to define the endothelial cell response to applied shear stress in vitro. A central question has been the mechanisms by which endothelial cells perceive and respond to changes in fluid flow. We have utilized cDNA microarrays to characterize the immediate/early genomic response to applied laminar shear stress (LSS) in primary cultures of human coronary artery endothelial cells (HCAECs). Cells were exposed, in a parallel plate flow chamber, to 0, 15, or 45 dyn/cm2 LSS for 1 h, and gene expression profiles were determined using human GEM1 cDNA microarrays. We find that a high proportion of LSS-responsive genes are transcription factors, and these are related by their involvement in growth arrest. These likely play a central role in the reprogramming of endothelial homeostasis following the switch from a static to a shear-stressed environment. LSS-responsive genes were also found to encode factors involved in vasoreactivity, signal transduction, antioxidants, cell cycle-associated genes, and markers of cytoskeletal function and dynamics.

Autocrine and paracrine effects of an ES-cell derived, BCR/ABL-transformed hematopoietic cell line that induces leukemia in mice.

During differentiation in vitro, Embryonic Stem (ES) cells generate both primitive erythroid and definitive myeloid lineages in a process that mimics hematopoiesis in the mammalian yolk sac. To investigate leukemic transformation of these embryonic hematopoietic progenitors, we infected differentiating cultures of ES cells with the Chronic Myeloid Leukemia-specific BCR/ABL oncoprotein. Following a period of liquid culture, we isolated two transformed subclones, EB57 and EB67, that retained characteristics of embryonic hematopoietic progenitors and induced a fatal leukemia in mice characterized by massive splenomegaly and granulocytosis. Histopathology of the spleen revealed an abundance of undifferentiated blast-like cells. Investigation of the clonal origins of the granulocytes in the peripheral blood demonstrated that the injected donor cells contributed modestly to the granulocyte population while the majority were host-derived. EB57 secretes IL-3 and unidentified cytokines that can stimulate autocrine and paracrine cell proliferation, presumably accounting for the reactive granulocytosis in diseased mice. These BCR/ABL transformed hematopoietic derivatives of ES cells recapitulate the relationship of BCR/ABL expression to IL-3 production that has been described for primitive hematopoietic progenitors from human CML patients, and illustrates the potential for autocrine and paracrine effects of BCR/ABL-infected cells in murine models.

Molecular anatomy of an intracranial aneurysm: coordinated expression of genes involved in wound healing and tissue remodeling.

BACKGROUND AND PURPOSE: Approximately 6% of human beings harbor an unruptured intracranial aneurysm. Each year in the United States, >30 000 people suffer a ruptured intracranial aneurysm, resulting in subarachnoid hemorrhage. Despite the high incidence and catastrophic consequences of a ruptured intracranial aneurysm and the fact that there is considerable evidence that predisposition to intracranial aneurysm has a strong genetic component, very little is understood with regard to the pathology and pathogenesis of this disease. METHODS: To begin characterizing the molecular pathology of intracranial aneurysm, we used a global gene expression analysis approach (SAGE-Lite) in combination with a novel data-mining approach to perform a high-resolution transcript analysis of a single intracranial aneurysm, obtained from a 3-year-old girl. RESULTS: SAGE-Lite provides a detailed molecular snapshot of a single intracranial aneurysm. These data suggest that, at least in this specific case, aneurysmal dilation results in a highly dynamic cellular environment in which extensive wound healing and tissue/extracellular matrix remodeling are taking place. Specifically, we observed significant overexpression of genes encoding extracellular matrix components (eg, COL3A1, COL1A1, COL1A2, COL6A1, COL6A2, elastin) and genes involved in extracellular matrix turnover (TIMP-3, OSF-2), cell adhesion and antiadhesion (SPARC, hevin), cytokinesis (PNUTL2), and cell migration (tetraspanin-5). CONCLUSIONS: Although these are preliminary data, representing analysis of only one individual, we present a unique first insight into the molecular basis of aneurysmal disease and define numerous candidate markers for future biochemical, physiological, and genetic studies of intracranial aneurysm. Products of these genes will be the focus of future studies in wider sample sets.

Activity of the farnesyl protein transferase inhibitor SCH66336 against BCR/ABL-induced murine leukemia and primary cells from patients with chronic myeloid leukemia.

BCR/ABL, the oncoprotein responsible for chronic myeloid leukemia (CML), transforms hematopoietic cells through both Ras-dependent and -independent mechanisms. Farnesyl protein transferase inhibitors (FTIs) were designed to block mutant Ras signaling, but they also inhibit the growth of transformed cells with wild-type Ras, implying that other farnesylated targets contribute to FTI action. In the current study, the clinical candidate FTI SCH66336 was characterized for its ability to inhibit BCR/ABL transformation. When tested against BCR/ABL-BaF3 cells, a murine cell line that is leukemogenic in mice, SCH66336 potently inhibited soft agar colony formation, slowed proliferation, and sensitized cells to apoptotic stimuli. Quantification of activated guanosine triphosphate (GTP)-bound Ras protein and electrophoretic mobility shift assays for AP-1 DNA binding showed that Ras effector pathways are inhibited by SCH66336. However, SCH66336 was more inhibitory than dominant-negative Ras in assays of soft agar colony formation and cell proliferation, suggesting activity against targets other than Ras. Cell cycle analysis of BCR/ABL-BaF3 cells treated with SCH66336 revealed G2/M blockade, consistent with recent reports that centromeric proteins that regulate the G2/M checkpoint are critical farnesylated targets of FTI action. Mice injected intravenously with BCR/ABL-BaF3 cells developed acute leukemia and died within 4 weeks with massive splenomegaly, elevated white blood cell counts, and anemia. In contrast, nearly all mice treated with SCH66336 survived and have remained disease-free for more than a year. Furthermore, SCH66336 selectively inhibited the hematopoietic colony formation of primary human CML cells. As an oral, nontoxic compound with a mechanism of action distinct from that of ABL tyrosine kinase inhibition, FTI SCH66336 shows promise for the treatment of BCR/ABL-induced leukemia.

Heterogeneous electron-transfer rate constants for M2(O2CR)4(0)/+, where M = Mo, W, Ru, or Rh and R = alkyl or aryl.

By the use of Nicholson's method, the heterogeneous electron-transfer rate constants (ks) for the oxidation of a series of M2(O2CR)4 complexes have been determined in benzonitrile, where the metal M = Mo, W, Ru, or Rh and R = alkyl or aryl. For R = tBu, the values of ks follow the order M = Mo > W > Ru > Rh. No simple influence of R on ks was observed, although added ligands that are known to reversibly bind to the dinuclear center were shown to influence the E1/2 values in order of their basicity and to suppress the rate of electron transfer. The reported data are compared with those obtained for Cp2Fe0/+, Cp2*Fe0/+, and Ru(bpy)2(2)+/3+ and with earlier work on dirhenium multiply bonded compounds.

Three novel activating mutations in the calcium-sensing receptor responsible for autosomal dominant hypocalcemia.

We report three novel activating mutations in the calcium-sensing receptor (CASR) that are responsible for autosomal dominant hypocalcemia (ADH) in three unrelated families. Each mutation involves a missense substitution resulting in a nonconservative amino acid alteration, P221L, E228Q, and Q245R. These mutations were observed in affected family members, but not in unaffected family members or in unrelated control samples. All three mutations are clustered in the extracellular domain of the CASR in a region dominated by negatively charged amino acids. Each mutant and wild-type receptor was expressed in Cos-1 cells. A luciferase reporter gene assay was utilized to detect the level of receptor activity by utilizing a protein kinase C-activated promoter to drive the production of luciferin, the reporter gene product. All three mutant receptors exhibited an increased sensitivity to calcium at all concentrations tested when compared to the wild-type receptor, supporting the hypothesis that these are activating mutations and are responsible for the ADH phenotype in these families. The data presented in this study suggest the importance of this highly negatively charged region of the extracellular domain in normal CASR function.

Functional polymorphism in the matrix metalloproteinase-9 promoter as a potential risk factor for intracranial aneurysm.

BACKGROUND AND PURPOSE: There is convincing evidence that susceptibility to intracranial aneurysms (ICAs) has a genetic component. However, few studies have sought to identify functional variation in specific candidate genes that may predispose individuals to develop an ICA. METHODS: ICA cases and controls were genotyped for a simple length polymorphism in the promoter of matrix metalloproteinase-9 (MMP-9) to test for association between variation in the promoter and the occurrence of ICA. Alternative alleles were cloned into an in vitro reporter vector, transfected into human HT1080 fibroblasts, and assayed for promoter activity by beta-gal and luciferase assays. Electrophoretic gel shift assays were used to assess nuclear factor binding. RESULTS: A length polymorphism in the promoter of MMP-9 was nonrandomly associated with the occurrence of ICA in a case-control study. This polymorphism was shown, by direct sequencing of 36 individuals, to be the only sequence variation within a 736-base pair region proximal to the transcriptional start site of the gene. Variation in the length of this repetitive element was shown to modulate promoter activity in an in vitro reporter assay, with the highest promoter activity being observed in constructs bearing the longest [(CA)23] element. Electrophoretic mobility shift assays were used to show that the (CA) element is bound by a sequence-specific DNA-binding protein. CONCLUSIONS: Genetic variation in the promoter of the MMP-9 gene results in variation in its expression at the level of transcription. This may result in subtle differences in MMP-9 activity within the circle of Willis, leading to increased susceptibility to ICA formation.

Comprehensive transcript analysis in small quantities of mRNA by SAGE-lite.

Serial analysis of gene expression (SAGE) is a powerful technique that can be used for global analysis of gene expression. Its chief advantage over other methods is that SAGE does not require prior knowledge of the genes of interest and provides quantitative and qualitative data of potentially every transcribed sequence in a particular tissue or cell type. Furthermore, SAGE can quantify low-abundance transcripts and reliably detect relatively small differences in transcript abundance between cell populations. However, SAGE demands high input levels of mRNA which are often unavailable, particularly when studying human disease. To overcome this limitation, we have developed a modification of SAGE that allows detailed global analysis of gene expression in extremely small quantities of tissue or cultured cells. We have called this approach 'SAGE-Lite'. This technique was used for the global analysis of transcription in samples of normal and pathological human cerebrovasculature to study the molecular pathology of intracranial aneurysms. These samples, which are obtained during operative surgical repair, are typically no bigger than 1 or 2 mm and yield <100 ng of total RNA. In addition, we show that SAGE-Lite allows simple and rapid isolation of long cDNAs from short (15 bp) SAGE sequence tags.

Gene expression is altered in perfused arterial segments exposed to cyclic flexure ex vivo.

Certain regions of coronary and other arteries undergo cyclic flexure due to attachment to the heart or crossing of joints. Such motion gives rise to fluctuations in transmural stress and luminal shear stress. It is well known that cyclic variation of these biomechanical forces influences many aspects of vascular cell biology including gene expression. The purpose of this work was to investigate the hypothesis that cyclic flexure of arterial segments influences their gene expression. Bilateral porcine femoral arteries were obtained fresh from the abattoir. One vessel was mounted in an ex vivo perfusion system and subjected to an intraluminal pressure of 60 mmHg and flow of 50 ml/min to serve as a control. The other vessel was mounted in a second perfusion system with similar hemodynamic conditions, but also subjected to controlled cyclic bending consistent with that found in coronary arteries in vivo. Reverse transcriptase-polymerase chain reaction analysis demonstrated that E-selectin and matrix metalloproteinase-1 (MMP-1) were consistently and significantly downregulated in the specimens subjected to 4 h of cyclic bending as compared to the control (n = 8, p < 0.05). Our results show that cyclic flexure of arterial segments in vitro may influence their gene expression. Further investigation should follow this novel observation and focus on other known mediators to more carefully elucidate the consequence of cyclic flexure on arterial pathobiology.

Unloading induces transcriptional activation of the sarco(endo)plasmic reticulum Ca2+-ATPase 1 gene in muscle.

Previous work showed that protein and mRNA levels of the "fast" isoform of the sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA1) are markedly increased in unloaded slow-twitch soleus muscles, suggesting pretranslational control of gene expression [L. M. Schulte, J. Navarro, and S. C. Kandarian. Am. J. Physiol. 264 (Cell Physiol. 33): C1308-C1315, 1993]. However, because of the difficulty of measuring transcription rates from whole muscle, transcriptional activation of the SERCA1 gene with unloading has not been confirmed. Because SERCA1 pre-mRNA levels can reflect transcriptional activity, in the present study SERCA1 introns were sequenced to allow intron-directed RT-PCR measurement of SERCA1 pre-mRNA. These data were then compared with changes in SERCA1 mRNA expression in control and unloaded soleus muscles. After 2, 4, and 10 days of unloading, SERCA1 pre-mRNA and mRNA transcript levels increased significantly by two-, three-, and sevenfold, respectively (P < 0.01). Parallel increases in SERCA1 pre-mRNA and mRNA suggest transcriptional activation of the endogenous SERCA1 gene by muscle unloading. SERCA2, the cardiac/slow-twitch skeletal muscle isoform, was not markedly increased by unloading, and RNase protection assays showed no change in alternative splicing of SERCA1 or SERCA2 primary transcripts. With use of in vivo plasmid injection, the activity of a reporter gene driven by 3.6 kb of the SERCA1 5'-flanking region increased fivefold in 7-day-unloaded soleus muscles. Comparison of the magnitude of transcriptional activation of endogenous and constructed SERCA1 genes by unloading confirms the fidelity of using intronic RT-PCR to examine muscle gene transcription rates and suggests that cis-acting elements sufficient for regulating unloading-induced transcriptional activation are contained in this promoter construct.

Neuroticism is not associated with the serotonin transporter (5-HTTLPR) polymorphism.

A deletion/insertion polymorphism in the transcriptional control region of the serotonin transporter gene (5-HTTLPR) was reported to be associated with dimensional measures of neuroticism, although subsequent replication attempts have failed. These replication attempts, however, have been dissimilar to the initial study in sample size, distribution of allelic frequency and/or assessment of neuroticism. The current study was conducted in a further attempt to replicate the initial finding using: (a) a sample that was more comparable to each of the individual samples in the initial report; and (b) identical psychometric methodology to assess neuroticism. Two hundred and twenty-five Caucasian adults were genotyped for the 5-HTTLPR polymorphism and completed the NEO Personality Inventory. Results did not replicate the association between the 5-HTTLPR polymorphism and neuroticism; individuals with the short form of this variant did not report higher NEO Neuroticism. Indeed, men with the short form reported lower Anxiety, a finding that is directionally opposite to the initial results. These findings, combined with other failures to replicate, indicate that the reproducibility of the association between the 5-HTTLPR polymorphism and neuroticism must be regarded as questionable. The contradictory findings suggest the need for a replication attempt in a large, normative sample that is stratified by ethnicity and sex.

Electrochemical behavior of 3-chloro-2,4-pentanedione in the presence of cobalt salen.

We have studied the catalytic two-electron reduction of 3-chloro-2,4-pentanedione by cobalt(I) salen electrogenerated at a glassy carbon cathode in acetonitrile containing tetramethylammonium tetrafluoroborate. When cobalt(I) salen is electrogenerated at -0.65 V (a potential that is 30 mV more negative than the peak potential for the reversible one-electron reduction of cobalt(II) salen), the carbon-chlorine bond of 3-chloro-2,4-pentanedione is catalytically cleaved to form 2,4-pentanedion-3-ate; this anion can be protonated either by adventitious water or by a deliberately added proton donor to produce 2,4-pentanedione, or the anion can be trapped with iodoethane to give 3-ethyl-2,4-pentanedione. However, when cobalt(I) salen is electrogenerated at -0.40 V (a potential at which the rate of generation of cobalt(I) salen is relatively small), the 2,4-pentanedion-3-ate, resulting from the catalytic two-electron cleavage described above, can deprotonate unreduced starting material to form 3-chloro-2,4-pentanedion-3-ate and 2,4-pentanedione. In further work, we have found that 2,4-pentanedion-3-ate can be oxidized directly to form the corresponding radical which couples to yield 3,4-diacetyl-2,5-hexanedione. Chemically produced 2,4-pentanedion-3-ate reacts with electrogenerated cobalt(III) salen to give a dionylcobalt(III) salen species which undergoes a one-electron reduction to liberate cobalt(II) salen and the dionate. In addition, cobalt(II) salen reacts with molecular oxygen to give cobalt(III) salen and superoxide, and the latter reduces 3-chloro-2,4-pentanedione to form chloride ion, the 2,4-pentanedion-3-yl radical, and molecular oxygen.

Skeletal muscle sarcoplasmic reticulum Ca(2+)-ATPase gene expression in congestive heart failure.

Congestive heart failure leads to skeletal muscle abnormalities, one of which is a prolongation of sarcoplasmic reticulum Ca2+ flux. The purpose of this study was to determine whether skeletal muscle of spontaneous hypertensive and heart failure rats have alterations in the expression of the sarcoplasmic (or endoplasmic) reticulum Ca(2+)-ATPase (SERCA) gene. Northern analysis revealed that SERCA1, the predominant skeletal muscle isoform, was decreased by 45%, 43%, and 58% in the tibialis anterior, plantaris, and diaphragm muscles, respectively. Ribonuclease protection assay showed that the decrease was due to the adult isoform, SERCA1a, with minor changes in the alternatively spliced neonatal isoform, SERCA1b. There was no change in SERCA1 mRNA levels in gastrocnemius muscles. No change was found in SERCA2a (cardiac/slow skeletal isoform) mRNA or protein levels or in SERCA2b (smooth muscle isoform), dihydropyridine receptor, or alpha-actin mRNA levels in diaphragm muscle. Northern blot and ribonuclease protection assays showed that SERCA2a decreased 61% in the heart while the alternatively spliced isoform, SERCA2b, decreased 27%. Western analysis of the tibialis anterior, diaphragm, and gastrocnemius muscles showed a decrease in SERCA1 protein levels by 46%, 64%, and 42%, respectively, whereas sarcoplasmic reticulum Ca(2+)-ATPase activity, a functional correlate of SERCA expression, was decreased by 38%, 38%, and 40% in the same muscles, SERCA2 protein expression decreased by 36% in the failing heart. Decreases in both mRNA and protein suggest pretranslational control of SERCA1 expression, whereas the lack of decreased SERCA1 mRNA in gastrocnemius muscle suggests translational regulation. The decreased SERCA1 protein expression in all muscles studied probably contributes to contractile abnormalities related to excitation-contraction coupling function in heart failure.

Genetic and molecular characterization of murine GATA-1 in Aspergillus defines a critical role for the N-terminal finger.

We have utilized Aspergillus nidulans as a model system for the characterization of the major vertebrate transcription factor GATA-1. This has been achieved both by analysing the function of murine GATA-1 directly and by using direct gene replacement to introduce chimaeric areA::GATA-1 derivatives at the areA locus, which encodes a GATA factor involved in regulating nitrogen metabolism in A. nidulans. Although GATA-1 shows only limited function when expressed in A. nidulans, the C-terminal GATA DNA-binding domain can replace the native GATA domain of AREA and retain near wild-type function. Surprisingly, inclusion of the N-terminal DNA-binding domain of GATA-1 has a major role in determining the function of areA::GATA constructs in vivo, leading to a general loss of activation. This negative function is partially dominant and is dependent on both the fidelity of the zinc-chelating structure and a second factor encoded by A. nidulans. The presence of two GATA domains also disrupts modulation of AREA activity. The ability of duplicate GATA domains to disrupt normal signal transduction is not dependent on the relative position of the domains or on the fidelity of the zinc-chelating structure. This demonstrates the utility of nitrogen metabolism's regulation in A. nidulans as a model system for the molecular and genetic characterization of heterologous GATA factors while also providing insights into native Aspergillus regulatory components.

Adaptation of the skeletal muscle calcium-release mechanism to weight-bearing condition.

In the present study, we examined whether weight-bearing condition can regulate the sarcoplasmic reticulum (SR) Ca(2+)-release mechanism. Measurements of alpha 1-subunit dihydropyridine (alpha 1-DHP) and ryanodine receptor levels were made in hypertrophied fast-twitch plantaris muscles 5 wk after surgical removal of synergist muscles (increased weight bearing) and in atrophied slowtwitch soleus muscles (14 days of non-weight bearing) of the rat. Rates of AgNO3-induced SR Ca2+ release were measured with fura 2 as the Ca2+ indicator and pyrophosphate as the precipitating ion during vesicular Ca2+ loading. Ca(2+)-release rates were 38, 49, and 58% lower in vesicles from hypertrophied vs. control muscles at AgNO3 concentrations of 0.05, 0.5, and 5 microM, respectively (control = 18.2 +/- 1.4 microM.mg-1. min-1). Western blots showed no differences in the relative expression of alpha 1-DHP or ryanodine receptor when IIID5 (monoclonal) or GP3 (polyclonal) antibodies were used. There was also no difference in ryanodine (10 nM) binding in Ca(2+)-incubated SR vesicles from hypertrophied muscles, suggesting no difference in the number of channels. In contrast, expression of alpha 1-DHP and ryanodine receptors was increased by 144 and 157% in non-weight-bearing soleus muscles, respectively. Scatchard analysis of DHP binding showed a 40% increase in maximum binding capacity and no change in the dissociation constant with non-weight-bearing muscles. The direction of modification of the SR Ca(2+)-release mechanism is opposite with increased and decreased weight bearing, but the mechanism by which this is achieved appears to be different.

Direct analysis of native and chimeric GATA specific DNA binding proteins from Aspergillus nidulans.

In Aspergillus nidulans the regulatory gene areA is responsible for mediating nitrogen metabolite repression. The areA product (AREA) represents an example of the GATA family of DNA binding proteins, which are characterised by the presence of a GATA domain consisting of a zinc finger within a highly conserved region of 52 amino acids. Among the other transcription factors included in this family is the principal erythroid transcription factor, GATA-1, which contains two GATA domains. In order to demonstrate high specificity binding of native AREA to DNA containing the sequence -GATA-, and investigate the presence in A.nidulans of other proteins with related specificities, we have used gel mobility shift assays. Both AREA-dependent and independent complexes have been identified. Two strains bearing chimeric genes were also characterised. In these, the region encoding the native GATA domain of AREA was replaced by sequences from murine GATA-1 cDNA encoding either the equivalent C-terminal domain or both the N and C-terminal domains. Strains bearing the areA::NC-GATA construct, which includes the sequence encoding both the N and C-terminal domains of GATA-1, leads to a pronounced increase in one of two AREA-dependent complexes and implicates the N-terminal domain of GATA-1 in mediating protein-protein interactions.

Skeletal muscle overload upregulates the sarcoplasmic reticulum slow calcium pump gene.

Functional data suggest that the kinetics of force production and relaxation are slowed in hypertrophied skeletal muscle because of chronic overload. The purpose of this study was to determine whether gene expression of the slow/cardiac isoform of the sarcoplasmic reticulum (SR) Ca(2+)-adenosinetriphosphatase (ATPase) pump is upregulated in overloaded fast-twitch plantaris muscles. Increased active muscle loading was induced in rat plantaris muscles bilaterally by surgical removal of gastrocnemius and soleus muscles. Mass of the plantaris muscle was 80% greater 5 wk after surgery than in age-matched unoperated control rats (P < 0.05). Expression of the slow pump mRNA was 135% greater in hypertrophied muscles, as determined from autoradiograms of Northern blots with use of a cDNA probe specific for the slow/cardiac isoform. A monoclonal antibody (7E6) was used to quantify slow Ca2+ pump in SR vesicles with use of Western blot analysis. Densitometry of blots showed that the relative expression of the slow pump protein was 130% greater in hypertrophied plantaris muscles. Expression of the fast SR Ca2+ pump protein isoform, assessed using monoclonal antibody A52, was 25% less in hypertrophied than in control muscles. The Ca2+ uptake rate and ATPase activity of SR vesicles was approximately 15% lower in hypertrophied plantaris muscles (P < 0.05). Differential phospholamban expression could not account for changes in SR Ca2+ handling, because it could not be detected in rat slow- or fast-twitch skeletal muscle.(ABSTRACT TRUNCATED AT 250 WORDS)